Filtered with samtools flag 1804 (samtools view -F 1804):
read unmapped (0x4)
mate unmapped (0x8, for paired-end)
not primary alignment (0x100)
read fails platform/vendor quality checks (0x200)
read is PCR or optical duplicate (0x400)
Fraction of mitochondrial reads (unfiltered BAM)
rep1
rep2
rep3
Rn = Number of Non-mitochondrial Reads
292681750
247276636
156369615
Rm = Number of Mitochondrial Reads
4207083
4134932
1470032
Rm/(Rn+Rm) = Frac. of mitochondrial reads
0.01417056666459395
0.016446864529320305
0.009313452151853837
SAMstat (filtered/deduped BAM)
rep1
rep2
rep3
Total Reads
147487386
159268434
102667862
Total Reads (QC-failed)
0
0
0
Duplicate Reads
0
0
0
Duplicate Reads (QC-failed)
0
0
0
Mapped Reads
147487386
159268434
102667862
Mapped Reads (QC-failed)
0
0
0
% Mapped Reads
100.0
100.0
100.0
Paired Reads
147487386
159268434
102667862
Paired Reads (QC-failed)
0
0
0
Read1
73743693
79634217
51333931
Read1 (QC-failed)
0
0
0
Read2
73743693
79634217
51333931
Read2 (QC-failed)
0
0
0
Properly Paired Reads
147487386
159268434
102667862
Properly Paired Reads (QC-failed)
0
0
0
% Properly Paired Reads
100.0
100.0
100.0
With itself
147487386
159268434
102667862
With itself (QC-failed)
0
0
0
Singletons
0
0
0
Singletons (QC-failed)
0
0
0
% Singleton
0.0
0.0
0.0
Diff. Chroms
0
0
0
Diff. Chroms (QC-failed)
0
0
0
Filtered and duplicates are removed.
Subsampling with atac.subsample_reads is not done in alignment steps.
Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled
with such parameter in the peak-calling step.
Fragment length statistics (filtered/deduped BAM)
rep1
rep2
rep3
Fraction of reads in NFR
0.9993395896975993
0.8476954749632342
0.9199384409576455
Fraction of reads in NFR (QC pass)
True
True
True
Fraction of reads in NFR (QC reason)
OK
OK
OK
NFR / mono-nuc reads
1882.6752072343631
5.803496499665853
11.768652859021797
NFR / mono-nuc reads (QC pass)
True
True
True
NFR / mono-nuc reads (QC reason)
OK
OK
OK
Presence of NFR peak
True
True
True
Presence of Mono-Nuc peak
False
False
False
Presence of Di-Nuc peak
True
False
False
rep1rep2rep3
Open chromatin assays show distinct fragment length enrichments, as the cut
sites are only in open chromatin and not in nucleosomes. As such, peaks
representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal)
fragment lengths will arise. Good libraries will show these peaks in a
fragment length distribution and will show specific peak ratios.
NFR: Nucleosome free region
Sequence quality metrics (filtered/deduped BAM)
rep1rep2rep3
Open chromatin assays are known to have significant GC bias. Please take this
into consideration as necessary.
Annotated genomic region enrichment
rep1
rep2
rep3
Fraction of Reads in universal DHS regions
0.36001564906710054
0.36051699359334444
0.3796856995035116
Fraction of Reads in blacklist regions
0.0009586650345813302
0.0016784430742880288
0.00201200254856773
Fraction of Reads in promoter regions
0.02446430232345429
0.02795852817891083
0.03228157220221455
Fraction of Reads in enhancer regions
0.3335907858587988
0.3302254481889362
0.34447562568313733
Signal to noise can be assessed by considering whether reads are falling into
known open regions (such as DHS regions) or not. A high fraction of reads
should fall into the universal (across cell type) DHS set. A small fraction
should fall into the blacklist regions. A high set (though not all) should
fall into the promoter regions. A high set (though not all) should fall into
the enhancer regions. The promoter regions should not take up all reads, as
it is known that there is a bias for promoters in open chromatin assays.
Library complexity quality metrics
Library complexity (filtered non-mito BAM)
rep1
rep2
rep3
Total Fragments
110773147
92609434
59293451
Distinct Fragments
75193012
80125720
51614861
Positions with Two Read
16055348
9296068
5782986
NRF = Distinct/Total
0.678802
0.8652
0.870499
PBC1 = OneRead/Distinct
0.684379
0.866793
0.871808
PBC2 = OneRead/TwoRead
3.205195
7.471163
7.781147
Mitochondrial reads are filtered out by default.
The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads
in a dataset; it is the ratio between the number of positions in the genome
that uniquely mapped reads map to and the total number of uniquely mappable
reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations
with EXACTLY one read pair over the genomic locations with AT LEAST one read
pair. PBC1 is the primary measure, and the PBC1 should be close to 1.
Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking,
0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is
the ratio of genomic locations with EXACTLY one read pair over the genomic
locations with EXACTLY two read pairs. The PBC2 should be significantly
greater than 1.
Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally
N1: Replicate 1 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep1 reads)
N2: Replicate 2 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep2 reads)
Ni: Replicate i self-consistent peaks (comparing two pseudoreplicates generated by subsampling RepX reads)
Nt: True Replicate consistent peaks (comparing true replicates Rep1 vs Rep2)
Np: Pooled-pseudoreplicate consistent peaks (comparing two pseudoreplicates generated by subsampling pooled reads from Rep1 and Rep2)
Self-consistency Ratio: max(N1,N2) / min (N1,N2)
Rescue Ratio: max(Np,Nt) / min (Np,Nt)
Reproducibility Test: If Self-consistency Ratio >2 AND Rescue Ratio > 2, then 'Fail' else 'Pass'
Number of raw peaks
rep1
rep2
rep3
Number of peaks
299456
299470
299458
The number of peaks is capped at 300000 Peaks are called from macs2 with p-val threshold 0.01
Peak calling statistics
Peak region size
rep1
rep2
rep3
idr_opt
overlap_opt
Min size
150.0
150.0
150.0
160.0
150.0
25 percentile
178.0
185.0
171.0
316.0
231.0
50 percentile (median)
211.0
245.0
221.0
448.0
324.0
75 percentile
301.0
369.0
332.0
630.0
475.0
Max size
2340.0
2457.0
2086.0
2452.0
3214.0
Mean
261.484181315452
305.6386950278826
277.94504738561
509.5519748263889
387.6935360540589
rep1rep2rep3idr_optoverlap_opt
Enrichment / Signal-to-noise ratio
Strand cross-correlation measures (filtered BAM)
rep1
rep2
rep3
Number of Subsampled Reads
12500000
12500000
12500000
Estimated Fragment Length
0
0
0
Cross-correlation at Estimated Fragment Length
0.17309035333131
0.182730706614741
0.180868064800158
Phantom Peak
40
65
50
Cross-correlation at Phantom Peak
0.176399
0.1687966
0.1692393
Argmin of Cross-correlation
1500
1500
1500
Minimum of Cross-correlation
0.15066
0.1597217
0.1594195
NSC (Normalized Strand Cross-correlation coeff.)
1.148881
1.144057
1.134542
RSC (Relative Strand Cross-correlation coeff.)
0.8714526
2.535462
2.18422
Performed on subsampled (25000000) reads.
Such FASTQ trimming is for cross-corrleation analysis only.
Fragment = read (for single-ended dataset) or pair of reads (for paired-ended dataset)
Normalized strand cross-correlation coefficient (NSC) = col9 in outFile
Relative strand cross-correlation coefficient (RSC) = col10 in outFile
Estimated fragment length = col3 in outFile, take the top value
rep1rep2rep3
TSS enrichment (filtered/deduped BAM)
rep1
rep2
rep3
TSS enrichment
3.122141923303737
6.3331474449049425
4.79470448688959
rep1rep2rep3
Open chromatin assays should show enrichment in open chromatin sites, such as
TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is
above 10. For other references please see https://www.encodeproject.org/atac-seq/
Jensen-Shannon distance (filtered/deduped BAM)
rep1
rep2
rep3
AUC
0.25765593542304766
0.2838827661987303
0.27447123443696314
Synthetic AUC
0.49628349055259524
0.4971632444397567
0.4962147028865357
X-intercept
0.15902905671167847
0.14314194816543388
0.15556768668102405
Synthetic X-intercept
0.0
0.0
0.0
Elbow Point
0.573317656186574
0.5444309135737296
0.5508937373398042
Synthetic Elbow Point
0.5066950327307322
0.5041903338140633
0.5064545074302387
Synthetic JS Distance
0.29386449490451394
0.2606668514302756
0.26577089471170084
Peak enrichment
Fraction of reads in peaks (FRiP)
FRiP for macs2 raw peaks
rep1
rep2
rep3
rep1-pr1
rep2-pr1
rep3-pr1
rep1-pr2
rep2-pr2
rep3-pr2
pooled
pooled-pr1
pooled-pr2
Fraction of Reads in Peaks
0.1449935725350777
0.13571182598555592
0.13294893586076625
0.14945795907647372
0.14044001788276492
0.1408635130463024
0.148297321484799
0.13921656489969086
0.14321965997927685
0.13472665218227411
0.1322271025998867
0.13088013991648104
FRiP for overlap peaks
rep1_vs_rep2
rep1_vs_rep3
rep2_vs_rep3
rep1-pr1_vs_rep1-pr2
rep2-pr1_vs_rep2-pr2
rep3-pr1_vs_rep3-pr2
pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks
0.061338501176392626
0.06644962955513647
0.06154695272365803
0.08175726295671143
0.07484930755330965
0.06262105662626928
0.08964799207682374
FRiP for IDR peaks
rep1_vs_rep2
rep1_vs_rep3
rep2_vs_rep3
rep1-pr1_vs_rep1-pr2
rep2-pr1_vs_rep2-pr2
rep3-pr1_vs_rep3-pr2
pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks
0.006553651188159653
0.011176371082511051
0.01875814550463644
0.020457173198526957
0.022380593005642286
0.01814295110187451
0.030807184719715358
For macs2 raw peaks:
repX: Peak from true replicate X
repX-prY: Peak from Yth pseudoreplicates from replicate X
pooled: Peak from pooled true replicates (pool of rep1, rep2, ...)
pooled-pr1: Peak from 1st pooled pseudo replicate (pool of rep1-pr1, rep2-pr1, ...)
pooled-pr2: Peak from 2nd pooled pseudo replicate (pool of rep1-pr2, rep2-pr2, ...)
For overlap/IDR peaks:
repX_vs_repY: Comparing two peaks from true replicates X and Y
repX-pr1_vs_repX-pr2: Comparing two peaks from both pseudoreplicates from replicate X
pooled-pr1_vs_pooled-pr2: Comparing two peaks from 1st and 2nd pooled pseudo replicates